In eukaryotes, many genes undergo alternative splicing and encode multiple isoforms leading to the expression of related proteins that have distinct biochemical as well as biological features. The IG20 (Insulinoma-Glucagonoma) is one such gene that undergoes alternative splicing and encodes at least four different splice variants (SVs), namely IG20pa, MADD/DENN, IG20-SV2 and DENN-SV. These four IG20-SVs are distinguished by differential splicing of exons 13L and 16. Upon comparison to IG20pa, the splice variants MADD, IG20-SV2 and DENN-SV lack the expression of exon 16 or 13L, or both respectively. All four IG20-SVs express an N-terminal leucine zipper and a C-terminal death-domain homology region.
The IG20 gene plays an important role in cancer cell proliferation, apoptosis and survival, most likely through its effects on MAP kinase activation and other cell signaling pathways. Additionally, it plays an important role in neurotransmission, neurodegeneration and guanine nucleotide exchange. How IG20 is involved in these divergent functions is not yet fully known.
Expression of the IG20 gene is relatively high in cancer cells and tissues as compared to the levels of expression in their normal counterparts. While MADD and DENN-SV are constitutively expressed (DENN-SV is over-expressed relative to other SVs in cancer), expression of IG20pa and IG20-SV2 appears to be regulated in that they may or may not be expressed in certain cells. Gain of function studies through expression of individual IG20-SVs in HeLa cells showed that MADD and IG20-SV2 variants have little or no effect on cell proliferation and induced apoptosis. IG20pa increased susceptibility to both extrinsic and intrinsic apoptotic stimuli, and suppressed cell proliferation and DENN-SV conferred resistance to induced apoptosis and enhanced cell proliferation. Thus IG20pa and DENN-SV acted like a “tumor suppressor” and an “oncogene” respectively.
Knock-down of all endogenous IG20-SVs, using anti-sense oligonucleotides, resulted in spontaneous apoptosis of cancer cells in vitro and in vivo, but not in normal cells. Since different splice variants have different functions and the function of IG20 gene may vary depending upon the cell type, it is prudent to develop modalities that allow knockdown of specific isoforms to achieve the desired effects including altering cell growth, apoptosis, neuron-transmission. Such isoform-specifc knock-down has not yet been demonstrated. In addition, the contrasting effects of IG20-SVs noted from gain of function studies can be clarified by knocking-down individual endogenous IG20-SVs and determining the consequent effects. This poses several challenges because various IG20-SVs differ from each other only by the differential expression of very short exons 13L (130 base pairs) and 16 (60 base pairs). Therefore, knock-down of specific splice variants of IG20 gene is difficult because of availability of very short target sequences that are differentially expressed in different splice variants and is achieved through the use of specially designed small hairpin RNA molecules (shRNA) disclosed herein.
Among the IG20 isoforms, MADD-SV acts as a negative regulator of caspase-8 activation and is necessary and sufficient for cancer cell survival. Abrogation of MADD-SV, but not the other IG20-SVs rendered cancer cells more susceptible to spontaneous as well as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. Also, the expression of MADD-SV alone in the absence of endogenous IG20-SVs is sufficient to prevent spontaneous apoptosis. MADD-SV plays a predominant role in cancer cell survival by acting as a negative regulator of caspase-8 activation. This profound effect is not due to direct association of MADD-SV with caspase-8. One possibility is that binding of MADD-SV to the receptor activates prosurvival pathways like MAP kinase or NF-kB pathway thereby antagonizing caspase-8 activation and leading to cancer cell survival.
The mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate several important and critical cellular functions required for cell homeostasis like metabolism, cell cycle progression, expression of cytokines, motility and adherence. Hence MAP kinases influence cell survival, proliferation, differentiation, development and apoptosis. Extracellular stimuli such as cytokines, growth factors and environmental stresses lead to the sequential activation of a signaling cascade composed of MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK. The three main members of MAPK family are extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun-amino-terminal kinase (JNK) and p38.
The ERK pathway is a drug target for cancer chemotherapy of all the mammalian MAPK pathways since in approximately, one-third of all human cancers there is deregulation of the pathway leading to ERK activation. When activated, ERK1/2 phosphorylates several nuclear and cytoplasmic substrates involved in a multitude of cellular processes, including transcriptional factors, signaling proteins, kinases and phosphatases, cytoskeletal proteins, apoptotic proteins and proteinases. Even though the ERK pathway can be activated by numerous extracellular signals, the pathways whereby cytokines and growth factors activate ERK signaling are of particular relevance to cancer. TNF-α, a cytokine rich in tumor stroma binds to TNFR1 (TNF receptor1) present on cancer cells and potently activates ERK MAPK. In the absence of MADD this pro-survival signaling pathway can be converted into an apoptotic signaling pathway leading to cancer cell death even in the absence of protein synthesis inhibitor like cycloheximide.
The mitogen-activated kinase activating death domain protein (MADD) is expressed at very low levels in a variety of tissues and organs under physiological conditions. However, it is over-expressed in many types of human tumors and tumor cell lines. Enforced expression of exogenous MADD has no apparent effect on cell survival, but knockdown of endogenous MADD can lead to spontaneous as well as enhanced tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) induced apoptosis, indicating that MADD is necessary for cancer cell survival. Furthermore, MADD contributes to the resistance of PA-1 ovarian carcinoma cells to TRAIL-induced apoptosis.
The extrinsic apoptotic pathway is initiated by upon death ligand (e.g. TRAIL), binding to its cognate death receptors, which undergo trimerization and recruit FADD and subsequent caspase-3 activation. MADD can bind to DRs thereby inhibit DISC formation. Knock-down of MADD causes spontaneous as well enhanced TRAIL-induced apoptosis. Thus, MADD can contribute to cell survival by blocking activation of the extrinsic apoptotic pathway.
The intrinsic pathway is initiated when a death signal induces the release of mitochondrial pro-apoptotic proteins such as cytochrome c, mitochondrial apoptosis-inducing factor and Smac/Diablo. Cytochrome c forms a complex with Apaf-1 and procaspase-9 resulting in the activation of caspase-9, while Smac/Diablo can associate with inhibitor of apoptosis proteins (IAPs) and counteract their inhibitory effects. The intrinsic pathway is regulated by the Bcl-2 family members. For example, in response to proapoptotic stimuli, the cytosolic Bax and Bad translocate to mitochondria leading to cytochrome c release into the cytosol. In contrast, Bcl-2 and Bcl-xL can associate with Bax and Bad to prevent cell death.
Apoptotic factors are controlled by a complex signaling system. For example, the pro-apoptotic function of Bad is regulated by phosphorylation that prevents Bad from inducing apoptosis. p53 function in regulating apoptosis is related to its phosphorylation status. The apoptosis repressor protein function is enabled by its phosphorylation at threonine-149. Given the important role of MADD in controlling apoptosis, there is a question whether its function is constitutive or is also regulated by other signals.
Protein kinase B (i.e. Akt) promotes cell survival by phosphorylating a variety of apoptosis-related factors containing the consensus sequence RXRXX(S/T). Akt phosphorylates mouse double minute 2 and enhances its ability to degrade p53. Akt can phosphorylate a number of apoptosis related proteins such as caspase-9, Bad, IKKα, Forkhead transcription factor, mdm2 and Yap, and play a critical role in TSC1/2, Rheb/mTOR signalling pathway. Interestingly, PI3K can also activate RAS function leading to the activation of the MAPK prosurvival pathway, in which MADD plays an important role.
Role of IG20 splice variants including MADD in cancer therapeutics was analyzed. Disruption of MADD phosphorylation by Akt results in death of cancer cells. siRNA molecules that specifically target IG20 splice variants are useful as pharmaceutical therapeutics against cancer.